Method of producing purified carotenoid compounds

ABSTRACT

A method of producing a carotenoid in solid form includes culturing a strain of Chlorophyta algae cells in a minimal inorganic medium and separating the algae comprising a solid form of carotenoid. In one embodiment f the invention, the strain of Chlorophyta algae cells includes a strain f  Chlamydomonas  algae cells.

STATEMENT OF GOVERNMENT INTEREST

[0001] Financial Assistance for this project was provided by the U.S.Government through the National Science Foundation under Grant No.DGE-9553456 and through the National Aeronautics and SpaceAdministration (NASA) under Grant No. NAG-W547. The United StatesGovernment may own certain rights to this invention.

FIELD OF INVENTION

[0002] The invention relates to methods for preparing carotenoids, andin particular, to the production of carotenoids using various strains ofalgae.

BACKGROUND OF THE INVENTION

[0003] It is now generally recognized in medical circles thatantioxidants play an important role in nutrition and in the preventionof certain diseases, such as macular degeneration of the eye.Accordingly, it is now routinely recommended that individuals consume a“recommended daily allowance” of antioxidants to maintain good health.

[0004] Among the important antioxidants are the carotenoids, of whichthe most commonly known is beta-carotene. Other common carotenoids arethe oxygenated form of beta-carotene, known as lutein, and anotheroxygenated carotenoid found in relatively low amounts in green plantsand algae, zeaxanthin.

[0005] While the carotenoids are known to have important health effects,these compounds are currently manufactured by expensive processesrequiring extraction of the compounds from a natural source, andsubsequent purification. As a result, for example, lutein, which is anabundant plant and algal carotenoid, is currently sold for about $50,000per gram. Zeaxanthin, produced from certain cyanobacteria, has a marketprice of approximately $100,000 per gram. These prices do not reflectscarcity, but rather the high cost of manufacture.

[0006] There is a great need for an inexpensive source of carotenoids topromote human health, such as the treatment of hives and otherdermatoses, and prevent certain types of eye disorders, such as maculardegeneration.

SUMMARY OF THE INVENTION

[0007] This summary of invention section is intended to introduce thereader to aspects of the invention and is not a complete description ofthe invention. Particular aspects of the invention are pointed out inother sections hereinbelow, and the invention is set forth in theappended claims which alone demarcate its scope.

[0008] The invention provides a unique method of preparing a carotenoid,zeaxanthin, through use of an alga. In particular, in one embodiment ofthe invention a Chlorophyta alga is cultured in a medium in the presenceof light, and then harvested. The harvested cells are separated, forexample, by centrifugation, and contain zeaxanthin and lutein, amongother components, in a solid product. The carotenoids are present in asufficient concentration in the solid for therapeutic or prophylacticadministration to humans, or may be further purified to increasecarotenoid concentration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The following drawings are illustrative of embodiments of theinvention and therefore do not limit the scope of the invention, but arepresented to assist in providing a proper understanding of theinvention. The drawings are not to scale, and are intended for use inconjunction with the explanations in the following detailed descriptionsection.

[0010]FIG. 1 is an output of a high pressure liquid chromatograph ofacetone extracted pigments recovered from a detergent-insoluble pellet,during an example in accordance with the invention;

[0011]FIG. 2 is an EDAX analysis of the pellet of FIG. 1, showing thepredominant inorganic component is phosphate, with divalent calcium andmagnesium cations;

[0012]FIG. 3 is a NMR spectrum showing that the polyphosphate is thepredominant form of the inorganic phosphate.

[0013]FIG. 4 is an SDS-PAGE of the detergent-insoluble pellet indicatingthat the major protein is a 70 kDa protein.

DETAILED DESCRIPTION OF THE INVENTION

[0014] This section describes aspects of the invention, and points outcertain preferred embodiments of these aspects. This section is notintended to be exhaustive, but rather to inform and teach the person ofskill in the art who will come to appreciate more fully other aspects,equivalents, and possibilities presented by the invention, and hence thescope of the invention as set forth in the claims which alone limit itsscope.

[0015] In accordance with an embodiment of the invention, a strain ofChlorophyta green algae is cultured under mild conditions in a minimalinorganic medium to produce cells that, upon harvesting, contain arelatively high concentration of carotenoids. The Chlorophyta algae mayinclude the genus Chlamydomonas, Dunaliella, or any other suitableChlorophyta algae. The medium may be formed of the following: 7.5 mMsodium acetate or sodium bicarbonate, 1.0 mM sodium citrate, 3.0 mMK₂PO₄, 7.0 mM KH₂PO₄, 7.5 mM NH₄Cl, 0.1 mM CaCl₂, 1.0 mM MgSO4, 0.01 mMFeCl₃ supplemented with 1 ml/l trace metals in the concentrationof(mg/100 ml) H₃BO₃, 100; ZnSO₄.7H₂O, 100; MnSO₄.4H₂O, 40; CoCl₂.6H₂O,20; Na₂MoO₄.2H₂O, 20; CuSO₄.5H₂O, 6. This description of the medium isnot to be limiting and it will be appreciated that any suitable mediumfor growing Chlorophyta may be used. The Chlorophyta algae cells aregrown in a temperature range of from approximately 25° C. to about 35°C. for a period of 3 to about 14 days. A detergent-insoluble pellet, forexample, a Triton-X 100-insoluble pellet, prepared from these cellscontains most of the zeaxanthin. The detergent insoluble pelletcontaining the algae cells may be extracted by suspending the pellet ina phosphate buffer, a Tris-HCL solution or any suitable suspendingmedium containing 1.0% (W/V) detergent, and sonicating and cetrifugingthe mixture. The pellet may then be prepared for use as a nutritionalsupplement by, for example, drying it and/or encapsulating.Alternatively, it may be subjected to heat to isolate the carotenoids.

[0016] In this example approximately 2 μg/mg of oxygenated carotenoidmay be isolated with the pellet (granule) preparation. This procedurecould apply to any carotenoid selected by spontaneous or engineeredmutation to be synthesized in Chlorophyta. For example, a strainproducing only zeaxanthin would yield a granular product containingzeaxanthin as the sole oxygenated carotenoid. Some carotenoid mutationsmay generate light sensitive strains of algae; however, due to theflexibility of Chlorophyta to growth conditions, the strain may be grownin the dark as long as a suitable carbon source such as acetate orglucose is provided. Stress, such as nutrient depravation, may alsostimulate synthesis and transfer of selected carotenoids to the granule.

EXAMPLE

[0017] Strain CC-373 (ac-u-c-2-21) used in this example was a gift froma stock maintained at the Chlamydomonas culture collection at DukeUniversity, Durham, N.C. Cells grown in a minimal inorganic medium at25° C. in the light for 3 to 4 days (late log to stationary phase) wereharvested by centrifugation at 1000 g. The medium was formed of thefollowing: 7.5 mM sodium acetate, 1.0 mM sodium citrate, 3.0 mM K₂PO₄,7.0 mM KH₂PO₄, 7.5 mM NH₄Cl, 0.1 mM CaCl₂, 1.0 mM MgSO₄, 0.01 mM FeCl₃supplemented with 1 ml/l trace metals in the concentration of (mg/100ml) H₃BO₃, 100; ZnSO₄.7H₂O, 100; MnSO₄.4H₂O, 40; CoCl₂.6H₂O, 20;Na₂MoO₄.2H₂O, 20; CuSO₄.5H₂O, 6. The pelleted cells were suspended in 50mM phosphate buffer (pH 7.0) or 10 mM Tris-HCl (pH 8.0) containing 1.0%(w/v) Triton X-100, broken by sonication and centrifuged at 1000 g for 5min. The pellet was washed two times with 1.0% Triton X-100 in buffer(50 mM phosphate, pH 7.0) and then two times with buffer. The resultingpellet contains zeaxanthin, lutein, polyphosphate, Ca²⁺, Mg²⁺, severalproteins and starch (FIGS. 2-4). Oxygenated carotenoids can be liberatedfrom the pellet fraction by a simple heating treatment.

[0018] Analysis of carotenoid composition of Chlamydomonas reinhardtiiCC-373 demonstrated that the strain was deficient in the epoxidatedcarotenoids neoxanthin (3S,5R,6R,3′S,5′R,6′S)-5′,6′-epoxy-6,7-didehydro-5,6,5′6′-tetrahydro-β,βcarotene-3,5,3′-triol),violaxanthin((3S,5R,6S,3′S,5′R,6′S)-5,6,5′,6′-diepoxy-5,6,5′,6′-tetrahydro-β,β-carotene-3,3′-diol,and antheraxanthin((3S,5R,6S,3′R)-5,6-epoxy-5,6-dihydro-β,β-carotene-3,3′-diol). In wholecells, the amount of zeaxanthin ((3R,3′R)-β,β-carotene-3,3′-diol) wasincreased to a level greater than that of lutein((3R,3′R,6′R)-β,ε-carotene-3,3′-diol). Upon analysis, only a relativelysmall amount of zeaxanthin was present in the membrane, which istraditionally considered its normal physiological domain. Rather, thebulk of zeaxanthin was recovered in a Triton X-100-insoluble pelletfraction (FIG. 1). The Triton X-100-insoluble fraction pelleted through1.5 M sucrose, a characteristic of polyphosphate granules that formwithin cytoplasmic vacuoles. Electron micrographs suggest that themembrane of these vacuoles is derived from the chloroplast envelope. Theformation of these dense cytoplasmic vacuoles is not confined toChlamydomonas but is thought to be common through Chlorophyta, i.e.Dunaliella, etc. The apparent transfer of zeaxanthin to chloroplastenvelope-derived cytosolic vacuoles allows for the simple preparation ofa zeaxanthin enriched product without the use of solvents.

[0019] The foregoing description provides an enabling disclosure of theinvention, which is not limited by the description, but only by thescope of the appended claims. All those other aspects of the invention,and their equivalents, that will become apparent when a person of skillin the art has read the foregoing, are within the scope of the inventionand of the claims hereinbelow.

I claim:
 1. A method of producing a carotenoid in a detergent-insolublepellet purified from soluble cellular components comprising: culturing astrain of Chlorophyta algae cells in a minimal inorganic growth medium;separating said algae cells from said minimal inorganic growth medium toobtain harvested algae; treating said harvested algae with detergent toobtain a detergent-insoluble pellet fraction comprising a carotenoid;and separating said detergent-insoluble pellet fraction from solublecellular material.
 2. The method of claim 1, wherein said strain ofChlorophyta algae cells comprises a strain of Chlamydomonas cells. 3.The method of claim 1, wherein said strain of Chlorophyta algae cellscomprises a strain of Chlamydomonas reinhardtii CC-373 cells.
 4. Themethod of claim 1, wherein the carotenoid is selected from zeaxanthinand lutein.
 5. A detergent-insoluble pellet form of carotenoid, thepellet form obtained from a process comprising: culturing a strain ofChlorophyta algae cells in a minimal inorganic growth medium; separatingsaid algae cells from said minimal inorganic growth medium to obtainharvested algae; treating said harvested algae with detergent to obtaina detergent-insoluble pellet fraction comprising a carotenoid; andseparating said detergent-insoluble pellet fraction from solublecellular material.
 6. The detergent-insoluble pellet form of carotenoidof claim 5, wherein said strain of Chlorophyta algae cells comprises astrain of Chlamydomonas cells.
 7. The detergent-insoluble pellet form ofcarotenoid of claim 5, wherein said strain of Chlorophyta algae cellscomprises a strain of Chlamydomonas reinhardtii CC-373 cells.
 8. Thedetergent-insoluble pellet form of carotenoid of claim 5, wherein thecarotenoid is selected from zeaxanthin and lutein.